Most proteins conform to the classical view that a polypeptide chain populates a single, stable native state. However, the phenomenon of fold switching, where protein sequences can exist at the interface between completely different folds, creates serious challenges to our understanding of how amino acid sequence encodes 3D structure. Additionally, it has many implications for understanding how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. Here, the overall objective is to determine experimentally how amino acid sequences migrate through fold space. We will determine the generality of fold switching and define its common principles by designing, engineering and analyzing a number of strategic protein switches. Our proposed studies employ small proteins that are widely used in experimental and computational folding studies, connecting our future results to a large body of knowledge. We aim to show that: 1) many folds can switch into other completely different topologies; 2) such switches can be designed/evolved; 3) structures and energetics of switches can be understood; 4) understanding can lead to prediction of other switches. Previous examination of both natural and engineered fold switches has shown that three conditions are generally necessary for a fold switch: 1) low stability of both folds; 2) compatibility of hydrophobic cores between folds; and 3) long range interactions in one fold which can override local interactions in the other. Methodical studies of fold switching require design and selection methods robust enough to create multiple examples of switches. To create different switches, we have chosen a series of origin folds that represent a range of common topologies (orthogonal bundle, 3-helix bundle, - grasp, SH3 barrel) that will be switched into different context-driven, destination folds (/ plait, / sandwich, Rossman-like). This approach allows us to satisfy the three general conditions of switching mentioned above. It also mimics evolutionary migration of sub-domains through fold space. Selection through the use of phage display methods will produce heteromorphic and bi-functional proteins that will be used for structural and energetic analysis. These proteins will be studied by a variety of physical methods including microcalorimetry, CD and NMR. Detailed structural and thermodynamic analysis will give important insights into the physicochemical basis for fold switching. The energetic and structural results will reveal how multiple folds are connected through short mutational pathways. These mutational connectivities in fold space will create networks of probable fold migrations. Our results will also enable computational biologists to use these data in folding simulations, fold network studies, and for further development and refinement of stability and structure prediction algorithms.